JP2511160B2 - Recombinant technology for producing novel natriuretic and vasodilator peptides - Google Patents

Abstract

The cDNA sequence encoding porcine brain natriuretic peptide and related genes encoding canine and human peptides with natriuretic activity are disclosed. The gene is shown to make accessible the DNAs encoding analogous natriuretic peptides in other vertebrate species. The genes encoding these NPs can be used to effect modifications of the sequence to produce alternate forms of the NPs and to provide practical amounts of these proteins. The NPs of the invention can also be synthesized chemically. The invention peptides have the formula: <tables id="TABLE-US-00001" num="00001"> <table frame="none" colsep="0" rowsep="0" tabstyle="monospace"> <tgroup align="left" colsep="0" rowsep="0" cols="2"> <colspec colname="1" colwidth="217pt" align="left"/> <colspec colname="2" colwidth="0pt" align="left"/> <TBODY VALIGN="TOP"> <ROW> <ENTRY>R<SUP>1</SUP>-Cys-Phe-Gly-Arg- Arg/ - Leu/ -Asp-Arg-</ENTRY> <ENTRY/> </ROW> <ROW> <ENTRY> Lys Met</ENTRY> </ROW> <ROW> <ENTRY> (1)</ENTRY> </ROW> <ROW> <ENTRY> Ile- Gly/ -Ser- Leu/ -Ser-Gly-Leu-Gly-Cys-R-<SUP>2</SUP></ENTRY> </ROW> <ROW> <ENTRY> Ser Ser</ENTRY> </ROW> </TBODY> </TGROUP> </TABLE> </TABLES> wherein R<SUP>1 </SUP>is selected from the group consisting of: <tables id="TABLE-US-00002" num="00002"> <table frame="none" colsep="0" rowsep="0" tabstyle="monospace"> <tgroup align="left" colsep="0" rowsep="0" cols="2"> <colspec colname="1" colwidth="217pt" align="left"/> <colspec colname="2" colwidth="0pt" align="left"/> <TBODY VALIGN="TOP"> <ROW> <ENTRY> (H);</ENTRY> <ENTRY/> </ROW> <ROW> <ENTRY> Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Ser-Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Asp/</ENTRY> </ROW> <ROW> <ENTRY> Lys/ -Ser-Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Gly</ENTRY> </ROW> <ROW> <ENTRY> Arg/ Asp/</ENTRY> </ROW> <ROW> <ENTRY> His/ - Lys/ -Ser-Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Gln Gly</ENTRY> </ROW> <ROW> <ENTRY> Arg/ Asp/</ENTRY> </ROW> <ROW> <ENTRY> Met/ - His/ - Lys/ -Ser-Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Val Gln Gly</ENTRY> </ROW> <ROW> <ENTRY> Arg/ Asp/</ENTRY> </ROW> <ROW> <ENTRY> Thr/ - Met/ - His/ - Lys/ -Ser-Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Met Val Gln Gly</ENTRY> </ROW> <ROW> <ENTRY> Arg/ Asp/</ENTRY> </ROW> <ROW> <ENTRY> Lys- Thr/ - Met/ - His/ - Lys/ -Ser-Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Met Val Gln Gly</ENTRY> </ROW> <ROW> <ENTRY> Arg/ Asp/</ENTRY> </ROW> <ROW> <ENTRY> Pro-Lys- Thr/ - Met/ - His/ - Lys/ -Ser-Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Met Val Gln Gly</ENTRY> </ROW> <ROW> <ENTRY> Arg/ Asp/</ENTRY> </ROW> <ROW> <ENTRY>Ser-Pro-Lys- Thr/ - Met/ - His/ - Lys/ -Ser-Gly-;</ENTRY> </ROW> <ROW> <ENTRY> Met Val Gln Gly</ENTRY> </ROW> </TBODY> </TGROUP> </TABLE> </TABLES> or a 10- to 109-amino acid sequence shown as the native upstream sequence for porcine, canine or human BNP in FIG. 8 , or a composite thereof; R<SUP>2 </SUP>is (OH), NH<SUB>2</SUB>, or NR'R'' wherein R' and R'' are independently lower alkyl (1-4C) or is <tables id="TABLE-US-00003" num="00003"> <table frame="none" colsep="0" rowsep="0" tabstyle="monospace"> <tgroup align="left" colsep="0" rowsep="0" cols="3"> <colspec colname="offset" colwidth="42pt" align="left"/> <colspec colname="1" colwidth="175pt" align="left"/> <colspec colname="2" colwidth="0pt" align="left"/> <TBODY VALIGN="TOP"> <ROW> <ENTRY/> <ENTRY>Asn/</ENTRY> <ENTRY/> </ROW> <ROW> <ENTRY/> <ENTRY>Lys</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Asn/ -Val</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Lys</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Asn/ -Val-Leu</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Lys</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Asn/ -Val-Leu-Arg</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Lys</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Asn/ -Val-Leu-Arg- Arg/</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Lys Lys</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Asn/ -Val-Leu-Arg- Arg/ - Tyr/</ENTRY> </ROW> <ROW> <ENTRY/> <ENTRY>Lys Lys His</ENTRY> </ROW> </TBODY> </TGROUP> </TABLE> </TABLES> or the amides (NH<SUB>2 </SUB>or NR'R'') thereof, with the proviso that if formula (1) is <tables id="TABLE-US-00004" num="00004"> <table frame="none" colsep="0" rowsep="0" tabstyle="monospace"> <tgroup align="left" colsep="0" rowsep="0" cols="3"> <colspec colname="offset" colwidth="21pt" align="left"/> <colspec colname="1" colwidth="196pt" align="left"/> <colspec colname="2" colwidth="0pt" align="left"/> <TBODY VALIGN="TOP"> <ROW> <ENTRY/> <ENTRY>R<SUP>1</SUP>-Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-</ENTRY> <ENTRY/> </ROW> <ROW> <ENTRY/> <ENTRY> Ile-Gly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R<SUP>2</SUP></ENTRY> </ROW> </TBODY> </TGROUP> </TABLE> </TABLES> and R<SUP>1 </SUP>is Asp-Ser-Gly-, R<SUP>2 </SUP>cannot be Asn-Val-Leu-Arg-Arg-Tyr. The peptides of the invention can be formulated into pharmaceutical compositions and used to treat conditions associated with high extracellular fluid levels, especially congestive heart failure.

Description

DETAILED DESCRIPTION OF THE INVENTION CROSS REFERENCE TO RELATED APPLICATION This is a US patent application No. 2 filed May 31, 1988.
This is a continuation-in-part application of US Patent Application No. 206,470 filed June 14, 1988, which is a continuation-in-part application of 00,383 1
This is a continuation-in-part application of US Patent Application No. 299,880, filed January 19, 989.

TECHNICAL FIELD The present invention relates generally to natriuretic and / or vasodilatory peptides found in brain and heart tissue. Further, the invention relates to genes encoding natriuretic and / or vasodilatory peptides obtained from porcine tissues, as well as genes encoding peptides related by amino acid sequences in other species. Furthermore, the present invention relates to a gene encoding a natriuretic and / or vasodilatory peptide from human atrium.

BACKGROUND ART It is well known that there are peptides in the atrium that maintain normal extracellular fluid parameters, namely the volume and pressure of fluid in blood vessels. A series of closely related peptides, termed atrial natriuretic peptides (atrial natriuretic peptides), have been isolated and identified from several species, and analogs of these peptides have been prepared. There is.

The natriuretic effect of the crude extract of rat atrial tissue was shown more than 7 years ago. A number of peptides with diuretic and natriuretic propeptides have since been isolated from atrial tissue and sequenced. Flynn, T.
G., et al., Biochem Biophys Res Commun (1983) 117 : 859-8.
65; Currie.MG, et al., Science (1984) 223 : 67-69; Kangaw
a, K., et al., Biochem Biophys Res Commun (1984) 118 : 131.
-139; U.S. Pat. No. 4,496,544; U.S. Pat. No. 4,508,712;
Kangawa, K., et al., Biochem Biophys Res Commun (1984) 1
19 : 933-940; Garcia, R., et al., Biochem Biophys Res Commu.
n (1985) 126 : 178-174; Katsube, N., et al., Biochem Biophy.
s Res Commun (1985) 128 : 325-330; U.S. Pat.No. 4,607,02
No. 3, 4,577,864; and 4,618,600; copending application 616,488; 766,030; and 870,795.
These peptides, called atrial natriuretic peptides (ANPs), are cyclic disulfides containing 17 amino acids in their ring, including two cysteines that provide disulfide bonds. The genes that encode them code for larger proteins, which
It is then processed into shorter proteins that make up the set of ANPs.

Various analogs of isolated atrial peptides have also been identified in co-pending applications 921,360; 138,893; and 174,73.
It is explained in No. 9.

These peptides and analogs thereof are understood to be effective in controlling blood pressure by controlling fluid volume and blood vessel diameter. Many pathologies are characterized by abnormal fluid retention, including congestive heart disease, cirrhosis, and nephrotic syndrome. These diseases are caused by the accumulation of excess fluid on the venous side of the circulatory system,
And associated with poor renal perfusion, which lead to decreased glomerular permeability (GFR). In addition, reduced renal perfusion stimulates renin secretion, proteolytic enzymes that lead to the formation of angiotensin in the circulatory system, and the powerful contractile muscles of small arteries. Renin also stimulates the release of the sodium-retaining hormone aldosterone from the adrenal glands.

Hypertension, by itself, is another serious consequence of increased extracellular fluid volume and is the leading cause of death.

Treatments for diseases associated with sodium and water retention are variable and include the administration of various diuretics. However, there are no therapeutic agents that satisfy all individuals, and it is important to expand the range of materials that can be used. In addition to supplementing a range of useful therapeutic agents, the present invention is important in that it is found in the brain and atria and sheds bright light on central and terminal functions, thereby maintaining a proper fluid balance in normal individuals. More substances. Furthermore, some of these peptides and proteins have altered and altered physiological activity.

One of these factors from the porcine brain is Sudoh, P., Natu
Re (1988) 332 ; 78-81 and isolated and sequenced. It is a 26 amino acid peptide synthesized in porcine brain and atrial tissue. In atrial tissue, the peptide is present at about 1 / 100th of the concentration of atrial natriuretic peptide (ANP) activity analyzed. However, in the brain, peptides may be more abundant than ANP. The spectrum of activity of this porcine brain natriuretic peptide or pBNP is similar to that of porcine ANP. A comparison of the amino acid sequences of the portions of human ANP (hANP) and pBNP is shown below. The corresponding relevant part of the corresponding porcine ANP is identical to the human sequence.

There are nine non-homologous parts (stars). The conservative substitution of Ile or Met for Leu found in rat or human ANP sequences is a known and acceptable substitution.

Subsequent papers from this same group at Miyazaki Medical College further characterized these proteins.
Sudoh, T., et al., Biochem Biophys Res Comm (1988) 155 : 7.
26-732 was derived from the porcine brain at the C-terminus by using the above-mentioned 26 Buda BNP.
Ser-Pro-, which is an additional sequence at the amino acid and N-terminus of
We have reported the isolation of a 32 amino acid natriuretic peptide ("BNP-32") containing a 6 amino acid extension called Lys-Thr-Met-Arg-.
The level of various natriuretic peptides in tissues is reported in a paper that continues on the next page. Ueda, S., et al., Ibid., Pp. 733-739, used a radioimmunoassay to localize and measure levels of porcine BNP and porcine BNP-32 in the brain and spinal cord. The results indicate that BNP and BNP-32 are the major forms of immunoreactive BNP in the porcine brain,
s), striatum and spinal cord were found to have the highest concentrations found. The porcine form of the atrial natriuretic peptide (pANP) was also found in pig brain at levels approximately 13-fold lower than that of BNP. Minamino, N., et al., Ibid., Pp.740-746, report the results of a radioimmunoassay of porcine BNP and ANP in terminal tissues. BNP concentrations were highest in the cardiac atria of the tissues assayed. The immunoreactive form of this protein is characterized primarily as a 12 kd high molecular weight form, accounting for 15% of all immunoreactive BNP in atrial tissue.
Less than is the low molecular weight form of pBNP or pBNP-32.

In a sequel to this publication, Minamino, N., et al., Biochem.
Biophys Res Comm (1988) 157 : 402-409 reported the isolation and characterization of this high molecular weight form of BNP from porcine heart. The complete amino acid sequence of this protein has been obtained and its carboxy terminus has 26 amino acids pBNP (and 32
Of the amino acid PBNP-32). The full-length protein contains 106 amino acids. Finally, Maekawa, K., et al., Ibid., PP.410-416, reported on the cloning and sequence analysis of the cDNA encoding the porcine BNP precursor protein. A cDNA library was obtained from porcine heart atrium and the relevant BNP-encoded gene was isolated and sequenced. The gene was found to contain a 25-residue putative signal peptide at the N-terminus, followed by codons corresponding to 106 amino acids of the reported protein.

These results are consistent with the information obtained from studies of atrial-derived natriuretic peptides, which are also commonly associated with longer precursors. In the parent application of this application,
A gene encoding porcine BNP has been provided, which predicted the deduced amino acid sequences of the upstream portions of these precursor proteins. CD obtained in this parent application
While the NA was incompletely processed and contained introns, further processing of this sequence using the standard techniques described below established the position of the intron. This parent application disclosed a direct method that can be applied to identify natriuretic-related peptide (NRP) from other species. The direct approach outlined did not clone human NRP. Furthermore, as explained below, it was not possible to obtain human NRP using only porcine BNP as a probe. In order to obtain human NRP, it was necessary to use an intermediate clone, dog BNP. Accordingly, the present invention provides a group of natriuretic peptides (NPs) and natriuretic-related peptides (NRPs) from various vertebrate sources.

DISCLOSURE OF THE INVENTION The present invention provides the complete gene sequence of pBNP, its prepro form, and the ability to synthesize large quantities of the protein encoded by this gene and its modified forms. The present invention also allows the recovery of gene sequences encoding similar proteins with natriuretic activity from other vertebrate species, as well as the ability to synthesize them. The cDNAs encoding porcine BNP, its precursor and shorter related brain proteins are shown in FIG. Sudoh (previous)
The "unprocessed" cDNA fragment encoding the 26 amino acid pBNP described by is underlined.

Accordingly, in one aspect, the present invention is directed to a recombinant cDNA probe comprising a sequence encoding a 26 amino acid natriuretic peptide of porcine brain, the recombinant cDNA probe comprising: DNA or its effective portion. The present invention is also directed to the recombinant DNA sequences recovered using this probe or a probe derived from this probe, and thus, alternatively, in the dog and human shown in FIGS. 3 and 5. Included is an effective probe containing an effective portion of the coding sequence of the peptide from the source.

In another aspect, the invention is directed to a peptide having natriuretic activity and having the formula: Where R ¹ is selected from the group Alternatively, it is the amino acid sequence from the 10th to the 109th, or a complex thereof shown in FIG. 8 as the natural upstream sequence of porcine, canine or human BNP; R ² is (OH), NH ₂ or NR. ′ R ″, wherein R ′ and R ″ are each independently lower alkyl (1-
4C), or Or their amides (NH ₂ or NR′R ″); provided that formula (1) is R ¹ -Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-Gly-Ser-Le.
When u-Ser-Gly-Leu-Gly-Cys-R ² and R ¹ is Asp-Ser-Gly, R ² is Asn-Val.
-Can't be Leu-Arg-Arg-Tyr.

In another aspect, the invention relates to recombinant DNA sequences encoding the above peptides, and recombinant expression systems capable of producing these peptides in appropriately transformed hosts. The invention also relates to a method of producing a peptide of the invention using recombinant means by culturing transformed cells and recovering the desired peptide from the cell culture.

The present invention is also directed to modified forms of this class of peptides, which contain one or two conserved amino acid substitutions.

The invention also relates to pharmaceutical compositions and methods of treatment using the peptides of the invention.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows porcine BNP with established intron position.
Figure 9 shows the complete sequence of the recovered cDNA in unprocessed form, which encodes The portion of the sequence encoding the 26 amino acid pBNP peptide is underlined and consists of nucleotide residues 660-723 and 1276-1289.

FIG. 2 shows an oligonucleotide synthesized as a probe of cDNA encoded by pBNP.

FIG. 3 shows the DNA and deduced protein sequences of the coding portion of the gene encoding the canine protein having sodium excretion enhancing activity.

Figure 4 shows human ANP (left panel) and canine NRP.
Shown is a Southern blot of human genomic DNA probed in (right panel).

FIG. 5 shows the DNA and deduced amino acid sequence of a human genomic clone encoding human NRP.

FIG. 6 shows a comparison of the amino acid sequences of the prepro forms of the porcine, canine and human proteins of the invention.

Modes for Carrying Out the Invention A. Definitions As used in the present application, "Brain Natriuretic Peptide (BNP) is an effective part of the DNA shown in Fig. 1 under defined strict conditions. Shows an amino acid sequence encoded by DNA that has the ability to hybridize to, and which has a natriuretic activity. All vertebrate brains contain a peptide similar to that disclosed in this application as pBNP. As used in the present application, which is believed to include a small population of peptides having, a longer precursor protein containing this amino acid sequence, and an active fragment thereof, "porcine brain natriuretic peptide (pBNP)" is , Isolated by Sudoh et al.
The 26 amino acid sequence presented above is shown. "CDNA encoding pBNP" refers to the nucleotide sequence shown in Figure 1 of the present application, including residues 660-723 and 1276-1289.
Separation of the pBNP codon in the cDNA may be due to incomplete processing of the mRNA that formed the template for this particular clone. This clone, 1988
It has been deposited with the American Type Culture Collection, Rockville, MD, on June 10, 2014, and its deposit number is ATCC 40465.

The pBNP-encoding cDNA shown in FIG. 1 contains an additional sequence encoding a precursor protein, as described below, and is therefore expressed by a sequence that separates the pBNP-encoding portion itself. To obtain genomic or cDNA sequences that may contain nucleotides corresponding to additional introns other than introns, which correspond in some other vertebrate species and which encode relevant brain natriuretic peptides. It can be used as an effective probe. As used in this application, "precursor brain natriuretic peptide" refers to a peptide having a natriuretic activity encoded by a gene sequence from which, for example, the pBNP protein is obtained. ， Processed to obtain peptides of different lengths. Similar processing differences may exist in other vertebrates. All classes of such natriuretic peptides are recovered by the DNA probes of the invention. For example, examination of the reading frame of the DNA encoding pBNP shows N-terminal extensions, such that N-terminally extended peptides can be postulated. ANP
The precursor, "proANP," is processed differently in atrial and brain tissues leading to different ANP peptides. By analogy with the peptides found in the atrium, the key terminal form of BNP is the tripeptide Thr-Met-Arg, which extends from the N-terminus to 29 of pBNP.
It seems to be a residue peptide. Additional upstream residues Ser-Pro-Lys and Gly-Ile-Arg-Ser-Pro-Lys are further N
Peptides extending from the end are also expected. Therefore, by examining the reading frame containing pBNP, it is possible to postulate additional upstream processing sites that would further extend the N-terminal sequence.

Other extended precursor peptides can be discovered by standard techniques using the sequence information in Figure 1. By analogy with the atrial natriuretic peptide precursor, the origin of the longest precursor that appears to contain the signal sequence is shown in the top reading frame of the line joining nucleotides 61 and 120 to the methionine codon. Or closely located downstream ATG
It is clear that it exists in. It is therefore clear that the reading frame is not maintained from the origin of this translation to the region encoding pBNP. This indicates that there is at least another intron transcribed in the recovered cDNA clone. The location of this intron and deduction of the full sequence of the longest form precursor peptide are described in more detail below. In any case, pBNP
The precursor BNP peptides related to Pt include other natriuretic peptides encoded by this depicted gene, and a similar group of peptides are collectively designated natriuretic peptides (NPs) , "Natriuretic peptide" (NRP) in other species
including.

Another useful term is the term "prepro" NRP, which is a naturally-occurring signal sequence that affects the secretion of various forms of peptides with natriuretic activity and a cyclic moiety that is absolutely necessary for this activity. Figure 3 shows an encoded peptide having the amino acid sequence of a secretory peptide fused upstream of. The "pro" form with the upstream sequence may represent the cyclic form of the peptide. Putative signals representing "pre" sequences for three specific embodiments encompassed by the present invention detailing porcine, canine and human proteins, respectively, in Figures 1, 3 and 5 The position of the sequence is shown in each figure along with the full-length mature protein, which is believed to be the precursor form indicating the "pro" form. Because of the variety of processing sites available, the clear distinction between "pro" NP and "NP" in the composite sequences shown in these figures and in Figure 6, as indicated by the up arrow, is meaningless. It appears to be. The peptides defined by the present invention are presented in formula (1) above, and N-, which precedes the identified 26 amino acid region corresponding to Sudoh's porcine "BNP", or which is linked to its cyclic portion. It has natriuretic and / or vasodilatory activity regardless of the length of the terminal shape.

"Expression system" refers to a DNA containing a coding region operably linked to appropriate control sequences capable of affecting its expression in a compatible host. The expression system always contains a promoter, but depending on the intended host, important additional DNA such as a ribosome binding site or a CAP site, and a termination sequence,
Enhancer sequences may be included, optionally upstream of the promoter or in other operable positions. Recombinant expression systems of the present invention include DNA of the present invention encoding BNP, eg, BNP obtained from vertebrate sources and operably linked to additional DNA sequences that can affect expression. Including. The expression system may be constructed so that it is present on a transfer vector, such as a plasmid or viral vector, which is self-replicating independently of the host cell's chromosome, or can be integrated into the chromosome when inserted into the host cell. .

B. Other Related Buda BNP and Vertebrate NRP Gene Recovery In one aspect, the invention provides all elements of the group of porcine BNP proteins encoded in the cDNA shown in FIG. 1 and their conserved forms. Regarding Deduction of the amino acid sequence encoding the longest precursor protein and deduction of the processed form is accomplished using the unprocessed cDNA provided herein. In this standard approach, an oligonucleotide sequence representing a potential intron, a short piece of cDNA linking the intron between residues 100 and about 660, is synthesized and
MRN labeled and isolated from cells producing BNP
Used to probe Northern blots of A.
Most mRNAs are in processed form. Thus, an oligonucleotide that successfully hybridizes to a messenger of the appropriate length represents the coding region of the cDNA. Oligonucleotides that do not readily hybridize represent intron regions. By using overlapping synthetic cDNAs, the position of the intron can be accurately identified. This allows estimation of the complete sequence encoding the largest precursor protein, limiting the sequences in which the associated BNP protein is formed.

In a variation of this approach, partial cDNA fractions were produced in amplified form from mRNA isolated from porcine atria. The cDNA for amplification was obtained by hybridizing poly A ⁺ RNA isolated from this tissue with oligonucleotide 3895. As shown in Fig. 1, the oligonucleotide primer has bases 100-1
23 (identified strand) and 652-685 (phase compensation). Amplification was performed using the polymerase chain reaction. However, analysis of the amplified product on a prepared agarose gel yields two bands. The larger band with lower relative abundance represents the smaller DNA from the unspliced precursor, and the higher abundance band is likely to be the more fully processed cDNA. When this band was eluted from the gel and sequenced, there was no extension corresponding to bases 223-468 in Figure 1 and the recovered DNA had the sequence shown in Figure 3.

Therefore, using standard techniques, Greenberg et al., Natu
The position of the putative upstream intron corresponding to the intron found in the atrial natriuretic peptide precursor was readily obtained, as described by re (1984) 312 : 656-658. As shown in Figure 1, a reading frame of 131 amino acids is obtained. The signal sequence is represented by amino acids 1 to 25, and the cleavage site that converts the prepro form of porcine BNP to the pro form is the site of the prepro sequence.
It is considered to be between Ser ₂₅ and His in. Sudoh
The sequence of a 26-amino acid porcine BNP reported by is represented by amino acid residues 81-106 (106-131).

In addition to providing access to the class of porcine BNPs encoded on the recovered cDNA, the cDNA of Figure 1 shows the corresponding precursors encoding classes of related NRP proteins from various vertebrate species. Provides access to genes.

The pBNP-encoding cDNA shown in Figure 1 or an effective portion thereof can be used as a probe in a gene library obtained from another vertebrate host by a number of techniques commonly known in the art. . Buda
There was every reason why BNP was considered to be able to hybridize to human NRP under appropriate conditions. However, the conditions under which pBNP hybridized to the human library could not be determined. From the perspective of evolution
It was surprising that porcine BNP appeared to be associated with the corresponding human protein rather than the corresponding canine protein.
Therefore, it was not expected that porcine BNP could not identify human NRP and that the canine gene needed to act as an intermediate.

The source of the desired gene may be a genomic library suitable for vertebrate species or a cDNA library from cells synthesizing peptides. As explained below, these peptides are synthesized in the brain but are also known to occur in atrial tissue at lower levels than normally produced ANP. Thus, the more easily accessible atrial tissue can also be used to prepare a cDNA library to be probed, rather than brain tissue. In this case, high stringency and +/− hybridization can be used to distinguish the more predominant ANP-encoding DNA.
It is well known in the art to prepare both genomic and cDNA libraries. In fact, some genomic libraries are commercially available. The preferred technique for preparing a cDNA library is Hyunh, VT, et al., DN.
A Cloning Techniques--A Practical Approach (IRL Pr
ess, Oxford, 1984) and Okayama and Berg, Mol Cel.
Biol (1983) 3 : 280-289. Preferably, for example, the method exemplified below is followed.

The genomic or cDNA library is then probed under non-stringent conditions to obtain hybridizing sequences. The recovered sequences can then be analyzed and sequenced according to standard procedures.

The entire DNA encoding pBNP in FIG. 1 can be used as a probe or the effective portion can be used. What constitutes an effective portion depends on the characteristics of the probed library and can be determined empirically. In general, when a genomic library is the source for recovery of the desired gene, a segment of the cDNA encoding pBNP extending from around residues 601-1300 is preferred. This segment binds an intron that interferes with the coding sequence of the pBNP protein. The upstream part of the sequence could also be used. Of course, there are no special drawbacks to using the whole clone. On the other hand, when the cDNA library is examined, it expresses the spliced coding region as usual
It is desired to use only a portion of the cDNA. Therefore,
For example, a suitable probe is the pBNP codon (parts 660-723).
And 1276-1289) flanking DNA or some smaller fragment thereof.

Of course, the actual probes can be the sequences shown, or preferably their complements.

In fact, the porcine DNA of Figure 1 can recover genes encoding related proteins in genomic libraries from other species, but not human NRP directly. Genomic libraries from pig, rat, dog, cat and rabbit showed the ability to hybridize to the probe of Figure 1 under at least one of the following conditions.

(1) 50% formamide, 6 x SSC, 5 x Denhardt, 10
mM sodium phosphate, 10 μg / ml sheared DNA at 42 ° C
And (2) 20% Holm Aodo, 6 x SSC, 5 x Denhardt, 10
mM sodium phosphate, 10 μg / ml shear DN at 37 ° C
A.

Wash under 1xS under both hybridization conditions
Sc, 0.1% SDS, 1 hour at 50-60 ℃.

Human genomic DNA did not hybridize to the DNA sequence of Figure 1 under these conditions, but it was possible to use canine NRP to obtain human NRP. Needless to say, the need to use the canine NRP sequence to obtain the human NRP gene sequence could not be expected.

By obtaining canine DNA using a porcine probe, an insert having the sequence shown in Fig. 3 was obtained, pdB
Designated as NP-1, it was deposited at the American Type Culture Collection on Dec. 14, 1988, with accession number ATCC-67862. Using this sequence as a probe, EcoRI
A clone was found, which was obtained from human genomic DNA digested with Escherichia coli and encodes a similar protein having a natriuretic activity. This human DNA had the sequence shown in FIG. 5, was designated as phBNP-1, and was deposited with the American Type Culture Collection on Dec. 14, 1988 under accession number ATCC-67863.

The amino acid sequence encoding the putative prepro form of the peptide with natriuretic activity from pig, dog and human species is shown in FIG. Pigs and dogs appear to be more homologous than the human sequences in the region presumably responsible for natriuretic activity. Using the information in Figure 6, the class of peptides with natriuretic activity can be identified. This class is shown by the following formula.

Where R ¹ is selected from the group Alternatively, it is the amino acid sequence from 10th to 109th, or its complex, shown in Fig. 6 as the natural upstream sequence of pig, dog or human BNP; R ² is (OH), NH ₂ or NR. ′ R ″, where R ′ and R ″ are each independently lower alkyl (1-
4C), or Or an amide thereof (NH ₂ or NR′R ″); provided that the formula (1) is R ¹ -Cys-Phe-Gly-Arg-Arg-Leu-Asp-Arg-Ile-G.
ly-Ser-Leu-Ser-Gly-Leu-Gly-Cys-R ² and R ¹ is Asp-Ser-Gly, R ² is Asn-Va.
It cannot be l-Leu-Arg-Arg-Tyr.

As used above, the “complex” of sequences shown in FIG. 6 as the native upstream sequence of porcine, canine or human BNP or NRP is the upstream sequence as shown therein. Sequences are shown in which each portion interchangeably comprises alternative amino acids shown in all three species. Complexes of these sequences are constructed like those for the sequences specifically shown in Formula (1) and the definitions of R ¹ and R ² above.

Furthermore, these peptide sequences can be modified by using one or two conservative amino acid substitutions, including substitutions using the D form rather than the L form, in place of specific moieties. These peptides can be synthesized using standard solid phase techniques, so it is not necessary, for example, to conserve conservative substitutions to be limited to the amino acids encoded by the gene.

Once the precursor genes encoding the BNP group of a particular vertebrate species have been recovered, the deduction of the BNP peptide associated with that species is a question of translation of the determined sequence and identification of processing sites. The pattern for the atrial natriuretic peptide counterpart provides guidance. By analogy with the atrial peptides, the BNP protein is believed to be a cyclic disulfide formed by the oxidation of cysteine residues at positions 4 and 20 of the pBNP sequence shown above. A class of encoded BNP peptides of a particular species is a truncated form outside this disulfide-bonded ring that contains one or two conserved amino acid substitutions in the sequence and the extension of pBNP shown. Morphology and.

The deduced (or generated) peptide sequence hybridizes in a buffer containing 20% formamide, 5 x Denhardt, 6 x SSC, 100 mg / ml RNA, and only 0.05% sodium pyrophosphate at 42 ° C. , Then 1 x SS
A DN encoding this peptide sequence under the conditions corresponding to the stringency indicated by washing with C, 0.1% SSD at 60 ° C or under the above conditions (1) or (2)
When A hybridizes directly or indirectly to the cDNA encoding pBNP of FIG. 1, it belongs to the range of the specific natriuretic protein of the present invention. Furthermore, this DN
The peptide encoded by A must exhibit a natriuretic activity assayed as described below.

"Direct hybridization" means that the DNA encoding the natriuretic peptide hybridizes to the DNA shown in FIG. 1 or its effective portion itself. "Indirect hybridization" means D
It means that NA hybridizes to DNA having the ability to hybridize to porcine BNP in FIG. Therefore,
The human sequence shown in FIG. 5 hybridizes indirectly to porcine BNP via the canine sequence in FIG. However, porcine BNP did not hybridize directly to human NRP.

The present invention also includes the BN encoded by the cDNA of FIG.
It relates to the denatured form of the P protein. One or two parts of these BNPs can be modified as long as the activity is retained. Conservative amino acid substitutions are preferred. That is, for example, aspartic acid / glutamic acid as acidic amino acid, lysine / arginine / histidine as basic amino acid, leucine / isoleucine / as hydrophobic amino acid.
Methionine / valine, and serine / glycine / alanine / threonine as hydrophilic amino acids. However, substitutions include non-encoded amino acids such as the D- or β-amino forms, as peptides do not need to be prepared by recombinant methods or from genes.

B. Preparation of BNP and NRP The BNP and NRP of the present invention can be prepared in a variety of ways, including the use of recombinant methods.

The recovered genes encoding the BNP and NRP peptides are then engineered and expressed using a variety of recombinant systems. Peptides having a sequence encoded by any subsegment of the recovered gene can be obtained by appropriately designing the expression system in a host system that does not process the translated protein. For example, an expression system may modify any flanking sequences appropriately to produce the desired N
It is constructed by placing an ATG start codon just before the end and a stop codon after the desired C-terminus. Then,
The desired coding sequence is optionally operably linked to a control system that functions in a prokaryotic or eukaryotic host. Many control systems are known in the art.

Since the natriuretic peptide precursor is clearly processed in certain eukaryotic systems, careful selection of recombinant hosts is necessary. Alternatively, processing can be prevented by modifying the gene sequence to encode an alternative amino acid at a position thought to be prone to cleavage by proteolytic enzymes. For example, you can replace the arginine immediately before the aspartic acid at position 1 of pBNP with a threonine residue,
As a result, the resulting peptide is insensitive to trypsin cleavage at that site. Alternatively, these peptides can be expressed in hosts lacking an enzyme capable of processing them.

Genes encoding a class of peptides associated with natriuretic activity in different vertebrate species are pBNP-encoding D
Probes constructed from NA are easily available, making it easier to obtain. Therefore, these genes can be engineered by replacing codons with one or more amino acids by site-specific mutations, resulting in sequences encoding analogs of these peptides that maintain natriuretic activity. it can.

Construction of expression vectors and production of recombinants derived from the appropriate DNA sequences are carried out by methods known per se in the art.

Expression can be performed in prokaryotic or eukaryotic systems. Prokaryotes are most often described by various strains of E. coli . However, Bacillus (eg, Ba
Cillus subtilis ), various strains of Pseudomonas , or other bacterial strains, such as strains of other bacteria, may also be used. In such prokaryotic systems, plasmid vectors containing replication sites and control sequences from species compatible with the host are used. For example, E.coli.
( Gene (1977) 2:95) and transformed with a derivative of pBR322, which is a plasmid derived from E. coli . Prokaryotic control sequences are commonly used and are defined herein to include promoters for initiation of transcription, optionally an operator, and a ribosome binding site sequence. It includes the β-lactamase (penicillinase) and lactose (lac) promoter systems (Chan
Et al., Nature (1977) 198 , 1056), tryptophan (tr
p) Promoter system (Goeddel et al., Nucleic Acids Res (198
0) 8: 4057), and P from lambda _L promoter and N-gene ribosome binding site (Shimatake et al., Nature (198
1) Commonly used promoters such as 292 : 128) are included. Any available promoter system compatible with prokaryotes can be used.

Expression systems useful in eukaryotic hosts include promoters from appropriate eukaryotic genes. The class of promoters useful in yeast includes, for example, promoters for synthesizing glycolytic enzymes. It includes a promoter for 3-phosphoglycerate kinase (Hitzeman et al., J Biol.
Chem (1980) 255 : 2073). Other promoters are derived from the enolase gene (Holland, MJ et al.,
J Biol Chem (1981) 256 : 1385) or derived from the Leu2 gene obtained from YEp13 (Broach, J. et al., Gene (1978)
8 : 121) is included.

 Suitable mammalian promoters include metallothionein, SV4
0-derived early or late promoters (Fiers et al.,Nature
(1978)273: 113), or polyoma, adenowi
Rus II, bovine papilloma virus or avian sarcoma virus
Includes other viral promoters, such as the traditional promoter
Is done. Appropriate viral and mammalian enhancers
The sensors are cited above. Plant cells as an expression system
If used, the nopaline synthesis promoter is suitable.
Yes (Depicker, A. et al.,J Mol Appl Gen(1982)1: 56
1).

Expression systems are constructed from the aforementioned regulatory sequences operably linked to BNP sequences by standard methods, using standard binding and restriction methods well understood in the art. The isolated plasmid, DNA sequence, or synthesized oligonucleotide is cleaved, trimmed, and recombined into the desired form.

Site-specific DNA cleavage is performed by treatment with an appropriate restriction enzyme (or multiple restriction enzymes) under conditions generally understood in the art. Certain of the above conditions are specified by the manufacturers of these commercially available restriction enzymes. For example, New England B
See iolabs, Product Catalog. In general, about 1 μg of plasmid or DNA sequence is cleaved with 1 unit of enzyme in about 20 μl of buffer solution; the examples herein are typically in excess to ensure complete digestion of the DNA substrate. Restriction enzymes are used. Although it can be changed, an incubation time of about 1 to 2 hours at about 37 ° C is adopted. After each incubation, proteins may be removed by extraction with phenol / chloroform followed by ether extraction. Then, the nucleic acid is recovered from the water-soluble fraction by precipitating with ethanol, and then applied to a Sephadex G-50 spin column. If desired, separation by size of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard methods. A general description of size separation is in Methods in Enzymology (198
0) 65 : 499-560.

The restriction fragment was 50 mM Tris (pH 7.6), 50 mM NaCl, 6 mM in the presence of 4 kinds of deoxynucleotide triphosphate (dNTP).
15 at 20-25 ° C in MgCl ₂ , 6 mM DTT and 5-10 μM dNTPs
Using incubation times of 25 minutes, E. coli. D
It can be made blunt-ended by treatment with a large fragment of NA polymerase I (Klenow). Klenow fragment is 5 '
It fills in the sticky end on the side, but even if four dNTPs are present, it sticks out the protruding single strand on the 3'side. If necessary,
Selective repair can be performed by supplying only one dNTP, the selected dNTP, within the limits dictated by the characteristics of the sticky ends. After treatment with Klenow fragment, the mixture is extracted with phenol / chloroform, ethanol precipitated and subsequently applied to a Sephadex G-50 spin column. Treatment with S1 nuclease under appropriate conditions hydrolyzes any single-stranded portion.

Synthetic oligonucleotides are prepared using commercially available automated oligonucleotide synthesizers. Single-chain kinase treatment prior to annealing or for labeling is 5 mM
Tris (pH7.6), 10mM MgCl ₂ , 5mM dithiothreitol, 1
~ 2mM ATP, 1.7pmole ³² P‐ATP (2.9mCi / mmole), 0.1mM
0.1 nmole in the presence of spermidine and 0.1 mM EDTA
Is performed using an excess (eg, about 10 units) of polynucleotide kinase over the substrate.

Couplings are performed in the following standard conditions and temperatures in a volume of 15-30 μl. That is, 20 mM Tris HCl (pH 7.5), 10 mM
MgCl ₂ , 10 mM DTT, 33 μg / ml BSA, 10 mM to 50 mM NaCl, and 40 μM ATP, 0.01 to 0.02 (Weiss) units of T4 DNA ligase at 0 ° C. (for “sticky end” ligation), or 1
mM ATP, 0.3-0.6 (Weiss) unit of T4 DNA ligase at 14 ℃
Or (for “blunt end” ligation). Intermolecular “sticky end” ligations are usually performed at 33-100 μg / ml total DNA concentration (5-100 nM total end concentration). Intermolecular blunt end ligation (usually with a 10-30 fold molar excess of linker) is performed at a final concentration of 1 μM.

In the construction of a vector using a "vector fragment", the vector fragment is usually treated with bacterial alkaline phosphatase (BA) in order to remove the 5'phosphate and prevent the vector from binding.
P). BAP digestion is carried out in the presence of Na ⁺ and Mg ⁺² with about 1 unit of BAP per μg of vector in about 150 mM Tris at pH 8,60 ° C. for about 1 hour. To recover the nucleic acid fragments, the preparation is extracted with phenol / chloroform, ethanol precipitated and desalted by running on a Sephadex G-50 spin column. Or 2
Recombination can be prevented by further digesting unwanted fragments of the digested vector with additional restriction enzymes.

Site-directed mutagenesis of primers is used for those parts of the vector from cDNA or genomic DNA that require sequence modifications. This is a single-stranded phage DNA to be differentiated
And with the exception of limited discrepancies, complementary primer synthetic oligonucleotides, which reveal the desired mutation. Briefly, synthetic oligonucleotides are used as primers to synthesize strands complementary to phage, and the resulting double-stranded DNA is transformed into phage-maintaining host bacteria. Cultures of transformed bacteria are plated on agar to form plaques from single phage-bearing cells.

Theoretically, 50% of the new plaques contain phage with the morphology mutated as a single chain and 50% have the original sequence. The obtained plaques are hybridized with the kinase-treated synthetic primer at a temperature at which the plaques are consistently hybridized but not hybridized with the original strand at a temperature at which the hybridization is sufficiently prevented. The plaque that hybridized with the probe is taken out, cultured, and the DNA is recovered. Details of the site-directed mutagenesis method are described in the specific examples below.

 The correct ligation for plasmid construction is firstE.col
i.Gene storage center (E.coli.Genetic Stock Cente
r),E.coli.Share MM294, CGSC # 6135, or other
Confirm by transforming the appropriate host with the ligation mixture.
Can be recognized. The successfully obtained transformants are
Ampicillin, tetrasa, as is understood in the field
Selected by resistance to Iclin or other antibiotics
Is done. The plasmid from this transformant was then transformed into C
lewell, D.B. et al.Proc Natl Acad Sci USA(1969)62: 11
59 method, followed by
Loramphenicol amplification (Clewell, D.B.,J Bacteriol
(1972)110: 667). The isolated DNA is
And / or Sanger, F. et al.Proc Natl Acad Sci USA
(1977)74: 5463, and more Messing et al.Nucleic Acids Res
(1981)9: 309 for sequencing by the dideoxy method
Or, alternatively, Maxam et al.Methods in Enzymology(198
0)65: 499 method.

The constructed vector is then transformed into a suitable host.

Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. Cohen, SN, Pro
c Calcium treatment with calcium chloride as described in Natl Acad Sci USA (1972) 69 : 2110, or
Maniatis et al., Molecular Cloning: A Laboratory Manual
(1982), Cold Spring Harbor Press, RbC described in p.254.
The l method is used for prokaryotic cells and other cells that have substantial cell wall barriers. Agrobacterium tumefaciens
Infection (Shaw, CH et al., Gene (1983) 23 : 315)
Is used for certain plant cells. For mammalian cells without such cell walls, Graham and van der Eb, Virolo
The calcium phosphate precipitation method of gy (1978) 52 : 546 is preferred. Transformation into yeast is described by Van Solingen, P. et al., J Bacter.
(1977) 130 : 946, and Hsiao, CL et al., Proc Natl Aca.
d Sci USA (1979) 76 : 3829.

The transformed cells are then cultured under conditions that favor expression of the BNP sequence, and the recombinantly produced protein is recovered from the culture.

In addition to recombinant production, peptides whose deduced sequences are short enough to allow direct peptide synthesis can be prepared using standard solid phase methods.

Thus, compounds within the scope of this invention may be chemically synthesized by methods well known in the art, such as solid phase peptide synthesis. The synthesis begins at the carboxy terminus of the peptide with an amino acid protected at the α-amino group. Even if other protecting groups are suitable, t-butyloxycarbonyl (Bo
c) Protecting groups can be used. For example, Boc-Val-OH, Boc-Leu
-OH, Boc-Arg-OH, or Boc-Tyr-OH (ie, the carboxy-terminal amino acid of selected BNP analogs) can be esterified to a chloromethylated polystyrene resin support. The polystyrene resin support has 0.5 to 2 as a cross-linking agent.
Copolymers of styrene with% divinylbenzene are preferred. The cross-linking agent renders the polystyrene polymer completely insoluble in the given organic solvent. Stewart et al., Solid-Phase
Peptide Synthesis (1969), WHfreeman, San Francisco, and Merrifield, J Am Chem Soc (1963) 8
See 5: 2149-2154. These and other methods of peptide synthesis are described in US Pat. No. 3,862,925; 3,84.
No. 2,067; No. 3,972,859; and No. 4,105,602.

Synthesis may be performed using the manual method, for example, Applied Biosystem 430A peptide synthesizer (Forster City, CA) or Biosearch SAM.
II An automated peptide synthesizer (Biosearch, San Rafael, Calif.) May be used to perform the procedure automatically according to the instructions provided by the manufacturer.

Of course, an automated synthetic method can also control the sequence, and thus the above modification can be used for the amino acid sequence obtained by modifying the gene as described above using this synthetic method. Furthermore, the substituted amino acids need not be genetically encoded. Thus, D-forms or β-amino acids can replace naturally occurring amino acids.

The above-described method of synthesizing the BNP of the present invention is not intended to be limited thereto, and the BNP of the present invention can be prepared by any conventional method. BNP is only required to be encoded by a gene that hybridizes to the cDNA of Figure 1 under the stringent conditions specified above, and to exhibit natriuretic activity in the receptor assay described below.

C. Assay Systems Members of the present invention of natriuretic peptides (NPs) and modifications thereof from various vertebrate species and origin are used to enhance the desired natriuretic activity using standard methods of assaying natriuretic activity. It can be confirmed to have activity. Numerous systems are available, both in vitro and in vivo. The simplest form of in vitro testing is in the kidney and other sites that affect the clearance of endogenous natriuretic compounds.
A binding assay that tests the affinity of peptides for receptors. Therefore, in a method similar to the assay for atrial-derived natriuretic peptides, general natriuretic activity was determined by the following results: cultured bovine aortic smooth muscle (BASM) cells and bovine aortic endothelium (BA).
E) pBNP labeled with a candidate peptide for binding to cell-derived receptors, eg by iodination
Can be analyzed by their ability to compete with. Competition is characteristic of binding to related clearance receptors. Moreover, the level of cyclic GMP can be measured in these same cells. And it is characteristic for the binding of the peptide to the relevant biological receptor responsible for the biological activity observed in vivo.

In order to fall within the scope of the composition claimed herein, the candidate peptide hybridizes directly or indirectly to the DNA encoding pBNP under the stringent conditions described herein. It must be encoded by the resulting gene sequence, or defined in formula (1), or a modification thereof as described above. In addition, the candidate peptide binds to the clearance receptor as shown by a competition assay in an in vitro receptor binding assay,
It must also exhibit either or both activity to binding to effector receptors as indicated by changes in cyclic GMP levels. The peptide
A peptide has direct biological activity associated with pBNP, such as cyclic GMP activity, if it inhibits clearance receptor binding in a manner related to in vivo testing for natriuretic and diuretic activity. You may or may not have it. The peptide can also be a vasodilator.

Receptor Binding Assays Specific ANP receptor sites have been identified in target tissues such as kidney, adrenal gland, blood vessels and cultured cells. Mapie
r, MA et al., Proc Nat Acad Sci USA (1984) 81 : 5946-594.
0: DeLean, A. et al., Endocrinology (1984) 115 : 1636-1638;
Schenk, DB et al., Biochem Biophys Res Comm (1985) 12
7 : 433-442. Such tissues have BNP-binding receptors. The receptor may or may not be the same as the receptor for ANP. ANP or A
Since it is hypothesized that binding of NP analogs to these specific receptor sites is a prerequisite for biological activity, B
Binding of NP-related peptides, or their modified forms, to these receptors is expected to predict biological activity.

Assays are generally based on Schenk (supra) and Scarborou
gh, RM et al., J Biol Chem (1986) 261 : 12960-12964. The Scarborough, RM disclosure evaluates the ability of ANP analogs to compete with labeled native ANP for binding to cultured BASM and BAE cells. A similar assay using labeled pBNP can be used to evaluate candidate BNP family peptides. pBNP (above) is iodinated at the carboxy terminal Y residue and is designated ( ¹²⁵ I) -pBNP. Similar "competitive displacement" receptor binding assays are considered in the art as a general case for testing specific ligand-receptor interactions.

In this assay, 0.5 nM ( ¹²⁵ I) -pBNP or (
¹²⁵ I) -human NRP in different amounts of unlabeled pBN
P, hNRP, dNRP or candidate peptide (cDN encoding pBNP
Encoded by a gene that hybridizes to A)
Incubate in individual samples of BASM cells.

Increasing concentrations of pBNP or successfully obtained candidate peptides effectively prevent binding of ( ¹²⁵ I) -pBNP to BASM cell associated receptors. ( ¹²⁵ I) -pBNP
The concentration of unlabeled peptide showing 50% of the maximum amount of binding was determined by Ki
It is called (app) and reflects the affinity of receptor binding.
Therefore, the peptide with Ki (app) = 100 nM is
= Interaction with the receptor, which is substantially weaker than the = 10 nM peptide. Assuming that these BNP analogs function at one or more receptor sites, increased receptor affinity should reflect increased biological potency.

Suitable natriuretic peptides for the competition assay described above should be selected in relation to the peptides of the invention tested. For example, the receptors used by ANP and BNP may be the same or different.
Any alternative species form can also be used as a competitor in the appropriate assay reaction for the candidate peptide of formula (1).

Systemic Mammalian Bioassays The biological activity of the NP sequences of the invention (which have shown activity in the receptor assay above) can be confirmed in anesthetized rats and dogs. Correlation of receptor binding affinity with in vivo efficacy is predictive of the receptor assay for biological activity.

1. Diuretic and Natriuretic in Anesthetized Rats One method involves placing a cannula in the left and right ureters and femoral veins of anesthetized rats and collecting urine from the ureters. The NP composition is administered through the femoral vein. Saline is infused for 30 minutes prior to NP infusion, urine is collected over 6 5-min baseline periods, and urine volume is determined gravimetrically.

After these baseline recovery periods, various NPs were infused for 30 or 60 minutes, and the volume of urine was increased over a period of 5 minutes during and 60 minutes after injection (while the rats were returned to saline). To measure. The data are evaluated by the urine flow rate averaged over six 5-minute baseline control periods immediately prior to infusion, comparing in-dose and post-dose values of NP with a "baseline" control value. The NP response is then calculated and plotted as a percentage of the baseline control response. Thus, the baseline ±
Responses to peptides above SD can be interpreted as a statistically significant increase.

2. Diuretic and Natriuretic Activity in Anesthetized Dogs The biological activity of the NPs of the present invention can be confirmed in dogs anesthetized with pentobarbital. In this example, a cannula is placed in the left and right ureters or bladders of the anesthetized dog and the femoral vein to collect urine. NP is given through the femoral vein. Saline before injection of NP
Infuse for 30 minutes and collect urine 3 times with a 10 minute collection period. Urine volume is determined gravimetrically and urine sodium content is determined spectrophotometrically.

After these three baseline collection periods, selected NPs will be infused for 60 minutes and urine rate and urinary sodium excretion will be measured for an additional 60 minutes after infusion. Injection (60 minutes) and recovery (60
Min), a collection period of 10 minutes is obtained. Control animals receiving saline only are tested in parallel.

Data are evaluated by comparing urine flow rate and sodium excretion rate in dogs infused with various NP moieties to control animals infused with saline.

Bioassay of Isolated Tissue The effect of NPs in vivo depends on receptor blockade (internal NPs).
(Including binding and removal of NPs) can be achieved alone by the ability to potentiate the effects of internal NPs. To the extent that this is the case for specific NPs, the diuretic and natriuretic effects of NPs are expected to be diminished or eliminated in isolated tissues where absent unless NPs were specifically supplied. To be done.

Thus, even though the activity of NPs in isolated tissue bioassays is low (although there is activity in these assays as well), NP compositions are within the scope of the invention.

D. Uses and Administration Briefly, the natriuretic peptides of the present invention are useful in the treatment of diseases such as hypertension, which are associated with high levels of extracellular fluid. This compound is Remi
ngton's Phermaceutical Siences , Mack Publishing, E
It is administered in conventional peptide formulations as described in Ston, Pa (latest edition). Preferably the peptide is
It is administered by injection, preferably intravenously, using a formulation suitable for this route of administration.
The dose level is about 0.01 to 100 μg / kg of subject.

Use of these compounds and compositions containing them as therapeutic agents for the treatment of various edema conditions in addition to hypertension and renal diseases caused by inefficient renal perfusion and decreased renal glomerular filtration rate Can be found. Such edema conditions include, for example, congestive heart failure, nephrotic syndrome and cirrhosis. The natriuretic peptide of the present invention is particularly effective for congestive heart failure.

Accordingly, the invention also provides compositions containing an effective amount of a compound of the invention. The compounds, including their non-toxic addition salts, amides, esters, alone provide the therapeutic benefits mentioned above. Such compositions may also be provided with physiologically acceptable liquid, gel or solid diluents, adjuvants and excipients.

These compounds and compositions can be administered to mammals in a manner similar to other therapeutic agents in veterinary applications such as farm animals, and in human clinical applications. Generally, the dose required for therapeutic effect is about 0 based on the body weight of the host.
It will be in the range of 001-100 μg / kg, more commonly 0.01-100 μg / kg. Alternatively, a dose within this range may be administered by constant infusion over a long period (usually over 24 hours) until the desired therapeutic effect is obtained.

Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; individuals suitable for solution or suspension in liquid prior to injection. Forms can also be prepared. The preparation may be emulsified. The active ingredient is often mixed with a diluent or excipient that is physiologically acceptable and compatible with the active ingredient. Suitable diluents and excipients are, for example, water, saline, glucose, glycerol and the like and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances. Auxiliary substances include wetting or emulsifying agents, stabilizing agents or pH buffering agents.

The composition is conventionally administered parenterally, for example by subcutaneous or intravenous injection. Alternative formulations that are suitable for other dosage forms include suppositories, intranasal aerosols, and optionally oral formulations. Traditional binders and excipients for suppositories include, for example, polyalkylene glycols or triglycerides; such suppositories have an activity within the range of 0.5-10%, preferably 1-2%. It may be formed from a mixture containing the components. Oral formulations include commonly used excipients such as pharmacological grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders, 10-95%, preferably 25-70%
Containing the active ingredient of.

The peptide compound may be formulated into the composition in neutral form or in salt form. Pharmacologically acceptable non-toxic salts include acid addition salts (formed by free amino groups) and inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as hydrochloric acid or phosphoric acid. Included are acids or salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid and the like. Salts formed with free carboxyl groups include, for example, inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxide, and isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine and the like. It can be derived from organic bases.

In addition to the compounds of the present invention that exhibit natriuretic, diuretic, or vasorelaxant activities, the compounds of the present invention can also be used as intermediates in the synthesis of such useful compounds. Alternatively, by appropriate selection, the compound of the invention, which has a reduced or completely abrogated activity level, is a diuretic, natriuretic or vasorelaxant compound (of the invention). Compounds outside the range are also included). Modulation of activity is performed, for example, by binding to clearance receptors, stimulating receptor turnover, providing another substrate for degrading enzyme or receptor activity, and inhibiting those enzymes or receptors. . When using this method, such compounds may be delivered as a mixture with other active compounds or may be delivered separately, eg in their own carrier.

The compounds of the present invention can also be used to prepare antisera for use in immunoassays using labeling reagents (usually antibodies). Polypeptides can be prepared using dialdehydes, carbodiimides, or commercially available linkers, if necessary,
Conveniently it can be bound to a carrier which renders it antigenic. These compounds and immunological reagents include various chromophores such as chromophores, fluorophores (eg fluorescein or rhodamine), radioisotopes (eg ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³ H) or magnetized particles. Labels may be labeled by methods well known in the art.

These labeled compounds and reagents, or labeled reagents that recognize them and can specifically bind to them, can find use as diagnostic reagents, for example. Samples from biological samples can be analyzed with the compounds of the invention for the presence or amount of substances with normal antigenic determinants. In addition, monoclonal antibodies can be prepared by methods known in the art, which antibodies find therapeutic use, eg, to neutralize in vivo overproduction of immunologically relevant compounds. Can be

Suitable subjects include animals with conditions that highly accumulate water or sodium ions. Both veterinary and human therapeutic applications are suitable.

The following examples are intended to be illustrative and not limiting of the invention.

Example 1 Recovery of DNA encoding porcine BNP A porcine heart tissue frozen in liquid nitrogen was obtained and roughly divided into an atrium and a ventricle. Frozen atrial tissue (5 g) was first crushed with a mortar and pestle, then powdered with a tissue crusher, 50 mM Tris (pH 7.5), 5 mM EDTA, 5
% Of β-mercaptoethanol (25 ml)
It was dissolved in 5M guanidine thiocyanate. Then sarcosyl was added to 2% and the sample was incubated at 65 ° C for 2 minutes. Then, the mixture was centrifuged at 7,000 xg for 10 minutes to remove insoluble substances. Then, 2.5 g of CsCl was added and overlaid on a 10 ml cushion of 5.8M CsCl, and again 2
Total RNA was isolated from this supernatant by centrifugation at 5,000 rpm for 12 hours. Subsequently, this supernatant was aspirated and the pellet was dissolved in a buffer solution containing Tris (50 mM), EDTA (5 mM) and β-mercaptoethanol (2%). Then, the RNA solution was once phenol extracted and precipitated with ethanol. Poly A ⁺ RNA was isolated by oligo dt cellulose chromatography.

Double-stranded DNA complementary to porcine atrial mRNA (5 μg)
Synthesized by the RNAse H method. The cDNA was then methylated by standard methods, ligated with EcoRI linkers and digested. The entire cDNA was then ligated into the previously prepared lambda phage arm and packaged by standard methods. About 1.75 x 10 randomly isolated from this library, which showed that almost all (> 95%) had inserts in the range of 1-5 kb by plating this packaging reaction. ^A library containing ⁵ phage was obtained.

A probe was designed to detect the pBNP cDNA. As shown in FIG. 2, the conservation of human ANP cDNA was estimated when oligo 3351 was constructed. Thus, the human ANP coding sequence shown in the figure was modified only to the extent necessary to obtain the pBNP coding sequence. The second oligo 3352 was another 60-mer designed according to the preference of mammalian codons favoring G and T over A and C. In addition, 60-mer 3376 is a human ANP to eliminate false positives.
Synthesized to fit the sequence.

Approximately 300,000 phage from the above library were plated and stripped in duplicate with a nitrocellulose filter. Series A (15 nitrocellulose filters) was denatured, neutralized and baked for 2 hours. And hybridization buffer (20% formamide, 5 x Denhard
solution, 6 x SSC, 0.05% pyrophosphate, 100 μg / ml salmon sperm DNA) for 2 hours. Next, labeled oligonucleotide probe (3351 1.5 x 10 ⁷
cpm) and the filters were incubated overnight at 42 ° C. Then, the filter was placed at 6 ° C for 20 minutes at 20 ° C.
It was washed in C, 0.1% SDS, then twice in 1 x SSD for 10 minutes at 65 ° C. Series B was treated similarly except that Oligo 3352 was not used. In this case, the final wash was at 60 ° C. In both cases, the filters were dried and autoradiographed. When probed with oligo 3351, about 450 positives (0.2% of all clones plated) were obtained. Most of these were based on previous screening with this oligo
Considered to be P. In series B, 4 clones hybridized to oligo 3352. These hybrids are stable at 60 ℃ but not stable at 65 ℃. Of these four, only clone 14 hybridized with oligo 3351. Then, this was taken out and purified again. The purified phage was then propagated. Then, the DNA was subjected to a CsCl step gradient of 36,000.
It was isolated by centrifugation at rpm, phenol extracted, dialyzed and ethanol precipitated. Ec for phage DNA
Restriction enzyme analysis with oRI contained a 1.5 kb DNA insert. The insert was then subcloned into the M13 sequencing vector and sequenced. The amount of this BNP mRNA is
About 400 times less than the ANP of this library, ie
It was 0.0005%.

The DNA sequence of the insert from clone 14 is shown in FIG. B
The coding region of NP is present in this clone. However, at the Val ₂₂ residue of the 26 amino acid BNP,
Suspended by what appears to be an intron. Therefore, this clone appears to contain unprocessed mRNA in which one or more introns are present.

Example 2 Identification of Upstream Introns As mentioned above, the DNA sequences in Table 1 show changes in reading frame. In addition, this DNA sequence
AN, as described by Greenberg et al. (Supra)
By analogy with the gene encoding P, it may contain an intron upstream. In order to determine the position of this intron, the sequence surrounding the putative position was used as a primer in the amplification method to obtain intron-free spliced DNA.

Poly A ⁺ RNA, Chirgwin, JM, Biochemistry (1979) 1
Isolation from porcine atrial tissue using the 8 : 5294-5299 guanidine isothiocyanate method followed by oligo-dT cellulose chromatography. About 2 μg of porcine atrial mRNA was added to 400 ng of oligonucleotide 3895.
Using the above (supra) as a primer, 0.5 mM DNTP and 50 mM Tri
s-HCl, pH 8.3, 10 mM magnesium chloride and 10 units RNa
The cells were cultured in 20 μl of reaction solution containing sin and 50 units of reverse transcriptase. Subsequently, the obtained DNA was subjected to Saiki, RK, Science
(1988) 239 : 487-491. After culturing at 37 ℃ for 1 hour, half of the reaction mixture was
M Tris-HCl, pH 8.8, 6.7 mM magnesium chloride, 16.6
mM ammonium sulfate, 10 mM mercaptoethanol, 6.7 μM EDTA, 1 mM dNTP, 10% DMSO, 400 ng
Each of the primer oligonucleotides was diluted to 100 μl. This oligonucleotide primer is composed of 100 to 123 bases (same strand) of the pBNP clone shown in FIG.
And 652 to 685 (complementary strands).

The reaction mixture was boiled for 5 minutes to denature it, followed by 42
Primer annealing was performed by incubating at ° C. Thermus aquaticus polymerase (3 units) was added and the sample was further incubated at 72 ° C for 3 minutes. This series of operations (98 ℃, 1min; 43 ℃, 1 minute; 72 ℃,
(3 minutes) was repeated 30 times without adding any more polymerase. Further reaction was allowed to occur under a 100 μl layer of mineral oil and 10 μl aliquots were removed and analyzed by standard agarose gel electrophoresis. Obtained DNA
To visualize the fragment of Stain with ethidium bromide,
Purified by electrophoresis using a prepared agarose gel. The amplified DNA fragment was then kinased, ligated into M13 and sequenced.

The two DNA sequences obtained from this reaction are as follows: That is, about 650 bp had a lower relative amount and was presumed to be unspliced. On the other hand, the more abundant band around 350 bp was considered to be fully processed DNA. In fact, this sequence shown in Figure 1 is found between the inverted arrows at positions 100-684.

Example 3 Recovery of gene encoding NRP All 1504 nucleotide sequences in FIG. 1, that is, bases 601-1300
The shorter segment, which composes, is used as a probe to obtain genes encoding similar NRP peptides of other vertebrate species. (However, as described in Example 5 below, human NRP could not be obtained by this direct method.) In this method, a blot of genomic DNA is used as a substrate. About 10 μg of genomic DNA from the liver of the appropriate species is digested with BamHI or PstI overnight. Then, the digested DNA was precipitated with ethanol and electrophoresed on a 0.8% agarose gel. The gel is then blotted overnight on a nitrocellulose filter. The filters are then denatured, baked, and at 42 ° C prehybridization buffer (20% formamide, 5 x Denhardt's 6 x SSC, 100 mg / ml RNA, 0.
Prehybridized in 05% sodium pyrophosphate).

The cDNA was labeled by nick-translation, and two panels from the same gel were hybridized at 42 ° C. overnight in prehybridization buffer. Then, these filters were used at 1 ℃ SSC, 0.1 ℃ at 60 ℃ and 65 ℃.
It was washed in% SDS and exposed to autoradiographic film.

The gene encoding a specific species of similar NRP was then amplified and sequenced by standard methods. The deduced sequence can be manipulated to provide suitable restriction sites for insertion into the expression system and site-directed mutagenesis to provide the desired stop and start codons. .

Southern blotting of genomic DNA from pig, rat, dog, cat, rabbit, and human organisms using the cDNA shown in Fig. 1 under the following two different hybridization conditions. Probed with: (1) 50% formamide, 6 x SSC, 5 x Denhardt, 10 mM
Sodium sulfate, 10 μg / ml sheared DNA, at 42 ° C; and (2) 20% formamide, 6 x SSC, 5 x Denhardt, 10 mM
Sodium sulfate, 10 μg / ml sheared DNA, at 37 ° C.

In all cases, the cleanliness was 5 x in 1 x SSC, 0.1% SDS.
It was performed at 0 to 60 ° C for 1 hour.

Hybridization did not occur between pBNP and human DNA.

Example 4 Recovery of canine NRP From the canine genomic library obtained from Clontech Inc., two clones were obtained under the condition (1) of Example 3 above. And of these identified clones
DNA was digested with HaeIII or AluI and subcloned into M13. The resulting plaques were screened for hybridization to porcine probes and the positive clones were sequenced. The identity of the clone was confirmed by detecting the sequence encoding BNP in FIG. Then, a 2.9 kb HindIII fragment containing the entire gene was added to pBR.
It was subcloned into 322 and named pdBNP-1. The DNA sequence of a portion of this clone encoding the BNP gene
Shown in the figure. The pdBNP-1 plasmid was assigned to the American Type Culture Collection on Dec. 14, 1988 under the accession number AT.
Deposited under CC-67862.

Example 5 Recovery of Human NRP As described in Example 3, the human genomic library could not obtain a signal corresponding to the hybridization with the probe using the porcine DNA shown in FIG. When pdBNP-1 was used as a probe under the condition (1) of Example 3 above, as shown in FIG. 4, several remarkable bands that could be visualized in the blot of digested human genomic DNA were obtained. Occurred. Preparative agarose gel was used to isolate human genomic DNA digested with EcoRI within the size range of 6-7 kb. And the isolated DNA is
It was cloned into λ-ZAP2 (Strategene Inc.) and packaged. The obtained mini library was screened using the hybridization condition of the condition (1) of the above example. Seven positive signals were purified and the insert was subcloned into pBLUSCRIPT vector. The M13 subclone of plasmid DNA digested with hybridization positive HaeIII and AluI was sequenced. The sequence of the coding region of plasmid phBNP-1 is shown in FIG. The plasmid was then deposited on Dec. 14, 1988 with the American Type Culture Collection under accession number ATCC-67863.

Using the intron splice junction consensus sequence described in Mount, S., Mucleic Acids Res (1982) 10 : 459-472, the first exon of the human cDNA sequence is located in the BNP precursor region relative to the porcine sequence. It seems to contain two extra amino acids. This can be confirmed by PCR amplification of human atrial RNA.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display location C12P 21/02 9162-4B C12N 15/00 ZNAA // (C12P 21/02 A61K 37/24 ABU C12R 1:19 ) ABR microbial accession number ATCC 67862 microbial accession number ATCC 67863 microbial accession number ATCC 40465 (72) Inventor Scarborough, Robert M. United States California 94544 Hayward, No. 2, Clearbrook Circle 29831 (72) Inventor Porter, Jay. Gordon United States California 94560 Newark, Dumas Place 7261 (56) Reference JP-A-63-107997 (JP, A) Nature, Vol. 332, P.I. 78-81 (1988)

Claims (7)

(57) [Claims] 1. A peptide having a sodium excretion enhancing activity and represented by the following formula: R ¹ -Cys-Phe-Gly-Arg-Lys-Met-Asp-Arg-Ile-Ser-Ser-Se.
r-Ser-Gly-Leu-Gly-Cys-R ² where R ¹ is selected from the following, Here, R ³ is a 102 amino acid sequence represented by the following formula: Or its C-terminal moiety; R ² is (OH), NH ₂ , NHR ′ or NR′R ″, where R ′ and R ″ are each independently lower alkyl (1-
4C) or R ² is Lys; Lys-Val; Lys-Val-Leu; Lys-Val-Leu-Arg; Lys-Val-Leu-Arg-Arg; Lys-Val-Leu-Arg-Arg. -His; or their amides (NH _2, NHR 'or NR'R "). 2. R ¹ is Ser-Pro-Lys-Met-Val-Gln-Gly-Ser-Gl
The peptide according to claim 1, which is y-, and R ² is Lys-Val-Leu-Arg-Arg-His or an amide thereof. 3. A pharmaceutical composition for inducing sodium excretion, diuresis and / or vasodilation in a subject, which comprises an effective amount of the peptide of claim 2 together with a suitable pharmaceutical excipient. The composition. 4. A recombinant expression system having the ability to express the DNA encoding the peptide of claim 1 when contained in a recombinant host cell. 5. A recombinant host cell or cell culture engineered to have the expression system of claim 4. 6. A method for producing a peptide having an activity of enhancing sodium excretion, which comprises culturing the cell according to claim 5 under conditions capable of expressing a DNA encoding the peptide; and a culture. Recovering said peptide from. 7. A DN encoding the peptide according to claim 1.
Recombinant DNA in isolated and purified form consisting essentially of A.